Our overall objective is to determine the relative contribution of tyrosinase synthesis, degradation, and activation to the MSH-induced increase in tyrosinase activity in melanoma cell cultures. Rates of tyrosinase synthesis and degradation will be determined by immunoprecipitation analysis of pulse-labeled enzyme. In addition we will quantitate the levels of tyrosinase-specific mRNA by in vitro translation assays and will correlate MSH-induced changes in tyrosinase mRNA levels with changes in enzyme synthesis rates and cellular tyrosinase activity. The presence of inactive tyrosinase in melanoma cells will be assessed through the use of competitive ELISA methodology and by immunoblot analysis. Further, we will utilize anti-tyrosinase IgG affinity chromatography to isolate both active and inactive forms of tyrosinase and will characterize their differences. Because of evidence from our laboratory which suggests that MSH promotes the production of a tyrosinase activator molecule, studies will be carried out to isolate this activator and to determine its mechanism of action. Studies to isolate and characterize an inhibitor of tyrosinase, which we have detected in these cells, will also be continued. (M)